The Ras proteins (Ha-Ras, Ki4a-Ras, Ki4b-Ras and N-Ras) are part of a signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein. In the inactive state, Ras is bound to GDP. Upon growth factor receptor activation Ras is induced to exchange GDP for GTP and undergoes a conformational change. The GTP-bound form of Ras propagates the growth stimulatory signal until the signal is terminated by the intrinsic GTPase activity of Ras, which returns the protein to its inactive GDP bound form (D. R. Lowy and D. M. Willumsen, Ann. Rev. Biochem. 62:851-891 (1993)). Mutated ras genes (Ha-ras, Ki4a-ras, Ki4b-ras and N-ras) are found in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. The protein products of these genes are defective in their GTPase activity and constitutively transmit a growth stimulatory signal.
Ras must be localized to the plasma membrane for both normal and oncogenic functions. At least three post-translational modifications are involved with Ras membrane localization, and all three modifications occur at the C-terminus of Ras. The Ras C-terminus contains a sequence motif termed a xe2x80x9cCAAXxe2x80x9d or xe2x80x9cCys-Aaa1-Aaa2-Xaaxe2x80x9d box (Cys is cysteine, Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willumsen et al., Nature 310:583-586 (1984)). Depending on the specific sequence, this motif serves as a signal sequence for the enzymes prenyl-protein transferase or geranylgeranyl-protein transferase, which catalyze the alkylation of the cysteine residue of the CAAX motif with a C15 or C20 isoprenoid, respectively. (S. Clarke., Ann. Rev. Biochem. 61:355-386 (1992); W. R. Schafer and J. Rine, Ann. Rev. Genetics 30:209-237 (1992)). The term prenyl-protein transferase may be used to refer generally to farnesyl-protein transferase and geranylgeranyl-protein transferase type I. The Ras protein is one of several proteins that are known to undergo post-translational prenylation. Other prenylated proteins include the Ras-related GTP-binding proteins such as Rho, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin. James, et al., J. Biol. Chem. 269, 14182 (1994), have identified a peroxisome associated protein Pxf which is also prenylated. These same authors have also suggested that there are prenylated proteins of unknown structure and function in addition to those listed above.
Inhibition of prenyl-protein transferase has been shown to block the growth of Ras-transformed cells in soft agar and to modify other aspects of their transformed phenotype. It has also been demonstrated that certain inhibitors of prenyl-protein transferase selectively block the processing of the Ras oncoprotein intracellularly (N. E. Kohl et al., Science, 260:1934-1937 (1993) and G. L. James et al., Science, 260:1937-1942 (1993)). Recently, it has been shown that an inhibitor of a prenyl-protein transferase blocks the growth of ras-dependent tumors in nude mice (N. E. Kohl et al., Proc. Natl. Acad. Sci U.S.A., 91:9141-9145 (1994)) and induces regression of mammary and salivary carcinomas in ras transgenic mice (N. E. Kohl et al., Nature Medicine, 1:792-797 (1995)).
Indirect inhibition of prenyl-protein transferase in vivo has been demonstrated with lovastatin (Merck and Co., Rahway, N.J.) and compactin (Hancock et al., ibid; Casey et al., ibid; Schafer et al., Science 245:379 (1989)). These drugs inhibit HMG-CoA reductase, the rate limiting enzyme for the production of polyisoprenoids including prenyl pyrophosphates. Prenyl-protein transferases utilizes prenyl pyrophosphates to covalently modify the Cys thiol group of the Ras CAAX box with a prenyl group (Reiss et al., Cell, 62:81-88 (1990); Schaber et al., J. Biol. Chem., 265:14701-14704 (1990); Schafer et al., Science, 249:1133-1139 (1990); Manne et al., Proc. Natl. Acad. Sci USA, 87:7541-7545 (1990)). Inhibition of prenyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane localization in cultured cells. However, direct inhibition of prenyl-protein transferases would be more specific and attended by fewer side effects than would occur with the required dose of a general inhibitor of isoprene biosynthesis.
Inhibitors of farnesyl-protein transferase (FPTase), a type of prenyl-protein transferase, have been described in two general classes. The first are analogs of farnesyl diphosphate (FPP), while the second class of inhibitors is related to the protein substrates (e.g., Ras) for the enzyme. The peptide derived inhibitors that have been described are generally cysteine containing molecules that are related to the CAAX motif that is the signal for protein farnesylation. (Schaber et al., ibid; Reiss et. al., ibid; Reiss et al., PNAS, 88:732-736 (1991)). Such inhibitors may inhibit proteinfarnesylation while serving as alternate substrates for the farnesyl-protein transferase enzyme, or may be purely competitive inhibitors (U.S. Pat. No. 5,141,851, University of Texas; N. E. Kohl et al., Science, 260:1934-1937 (1993); Graham, et al., J. Med. Chem., 37, 725 (1994)). In general, deletion of the thiol from a CAAX derivative has been shown to dramatically reduce the inhibitory potency of the compound. However, the thiol group potentially places limitations on the therapeutic application of FPTase inhibitors with respect to pharmacokinetics, pharmacodynamics and toxicity.
It has recently been reported that prenyl-protein transferase inhibitors are inhibitors of proliferation of vascular smooth muscle cells and are therefore useful in the prevention and therapy of arteriosclerosis and diabetic disturbance of blood vessels (JP H7-112930).
It has recently been disclosed that certain tricyclic compounds which optionally incorporate a piperidine moiety are inhibitors of FPTase (WO 95/10514, WO 95/10515 and WO 95/10516). Imidazole-containing inhibitors of farnesyl protein transferase have also been disclosed (WO 95/09001 and EP 0 675 112 A1).
It is, therefore, an object of this invention to develop compounds that will inhibit prenyl-protein transferase and thus, the post-translational prenylation of proteins. It is a further object of this invention to develop chemotherapeutic compositions containing the compounds of this invention and their methods of use.
The present invention comprises piperazine-containing macrocyclic compounds which inhibit prenyl-protein transferase. Further contained in this invention are chemotherapeutic compositions containing these prenyl transferase inhibitors and their methods of use.
The compounds of this invention are illustrated by the formula A: 
The compounds of this invention are useful in the inhibition of prenyl-protein transferase enzymes, such as farnesyl-protein transferase which is responsible for the farnesylation of the oncogene protein Ras. A first embodiment of this embodiment of this invention is illustrated by a compound of formula A: 
wherein:
R1a, R1b, R1c and R1d are independently selected from:
1) hydrogen,
2) aryl,
3) heterocyclyl,
4) C3-C10 cycloalkyl,
5) C2-C6 alkenyl,
6) C2-C6 alkynyl,
7) OR10,
8) R11S(O)mxe2x80x94,
9) R10C(O)NR10xe2x80x94,
10) (R10)2Nxe2x80x94C(O)xe2x80x94,
11) CN,
12) NO2,
13) (R10)2Nxe2x80x94C(NR10)xe2x80x94,
14) R10C(O)xe2x80x94,
15) R10OC(O)xe2x80x94,
16) N3,
17) xe2x80x94N(R10)2,
18) R11OC(O)NR10xe2x80x94, and
19) C1-C6 alkyl, said alkyl optionally substituted with one or more substituents selected from the following:
a) aryl,
b) heterocyclyl,
c) C3-C10 cycloalkyl,
d) C2-C6 alkenyl,
e) C2-C6 alkynyl,
f) R10Oxe2x80x94,
g) R11S(O)mxe2x80x94,
h) R10C(O)NR10xe2x80x94,
i) (R10)2Nxe2x80x94C(O)xe2x80x94,
j) CN,
k) (R10)2Nxe2x80x94C(NR10)xe2x80x94,
l) R10C(O)xe2x80x94,
m) R10OC(O)xe2x80x94,
n) N3,
o) N(R10)2, or
p) R11OC(O)xe2x80x94NR10xe2x80x94;
R2 is selected from:
1) oxo,
2) C1-8 alkyl,
3) C2-8 alkenyl,
4) C2-8 alkynyl,
5) aryl,
6) heterocyclyl,
7) (CO)NR6R7, and
8) (CO)OR6,
xe2x80x83said alkyl, alkenyl, alkynyl, aryl and heterocyclyl is optionally substituted with one or more of the following:
1) aryl or heterocyclyl, optionally substituted with:
a) C1-4 alkyl,
b) (CH2)pOR6,
c) (CH2)pNR6R7,
d) halogen, or
e) CN,
2) C3-6 cycloalkyl,
3) OR6,
4) S(O)mR4,
5) NR6R7,
6) NR6(CO)R7,
7) NR6(CO)NR5R7,
8) O(CO)NR6R7,
9) O(CO)OR6,
10) (CO)NR6R7,
11) SO2NR6R7,
12) NR6SO2R4,
13) (CO)R6,
14) (CO)OR6,
15) N3, and
16) F, or
xe2x80x83two R2""s are attached to the same C atom and are combined to form xe2x80x94(CH2)uxe2x80x94 wherein one of the carbon atoms is optionally replaced by a moiety selected from: O, S(O)m, xe2x80x94NC(O)xe2x80x94, and xe2x80x94N(COR10)xe2x80x94;
R4 is selected from:
1) C1-6 alkyl,
2) C3-6 cycloalkyl,
3) heterocyclyl, and
4) aryl,
xe2x80x83said alklyl, cycloalkyl, and heterocyclyl optionally substituted with one or more of the following:
a) C1-4 alkoxy,
b) aryl,
c) heterocyclyl,
d) halogen,
e) OH,
f) (CO)R11,
g) SO2R11, or
h) N(R10)2;
R5, R6 and R7 are independently selected from:
1) H,
2) C1-6 alkyl,
3) C3-6 cycloalkyl,
4) heterocyclyl,
5) aryl,
6) aroyl,
7) heteroaroyl,
8) arylsulfonyl, and
9) heteroarylsulfonyl,
xe2x80x83said alkyl, cycloalkyl, heterocyclyl, aryl, aroyl, heteroaroyl, arylsulfonyl, and heteroarylsulfonyl is optionally substituted with one or more of the following:
a) C1-4 alkoxy,
b) aryl,
c) heterocyclyl,
d) halogen,
e) OH,
f) (CO)R11,
g) SO2R11, or
h) N(R10)2,
R6 and R7 may be joined in a ring, and independently,
R5 and R7 may be joined in a ring;
R8 is selected from:
1) aryl,
2) heterocyclyl,
3) C3-C10 cycloalkyl,
4) C2-C6 alkenyl,
5) C2-C6 alkynyl,
6) perfluoroalkyl,
7) halogen,
8) R10Oxe2x80x94,
9) R11S(O)mxe2x80x94,
10) R10C(O)NR10xe2x80x94,
11) (R10)2NC(O)xe2x80x94,
12) R102Nxe2x80x94C(NR10)xe2x80x94,
13) CN,
14) NO2,
15) R10C(O)xe2x80x94,
16) R10OC(O)xe2x80x94,
17) N3,
18) N(R10)2,
19) R11OC(O)NR10xe2x80x94, and
20) C1-C6 alkyl, said alkyl optionally substituted with the following:
a) aryl,
b) heterocyclyl,
c) C3-C10 cycloalkyl,
d) C2-C6 alkenyl,
e) C2-C6 alkynyl,
f) perfluoroalkyl,
g) halogen,
h) R10Oxe2x80x94,
i) R11S(O)mxe2x80x94,
j) R10C(O)NR10xe2x80x94,
k) (R10)2NC(O)xe2x80x94,
l) R102Nxe2x80x94C(NR10)xe2x80x94,
m) CN,
n) R10C(O)xe2x80x94,
o) R10OC(O)xe2x80x94,
p) N3,
q) xe2x80x94N(R10)2, and
r) R11OC(O)NR10xe2x80x94;
R9 is selected from:
1) C2-C6 alkenyl,
2) C2-C6 alkynyl,
3) perfluoroalkyl,
4) halogen,
5) R10O,
6) R11S(O)mxe2x80x94,
7) R10C(O)NR10xe2x80x94,
8) (R10)2NC(O)xe2x80x94,
9) R102Nxe2x80x94C(NR10)xe2x80x94,
10) CN,
11) NO2,
12) R10C(O)xe2x80x94,
13) R10OC(O)xe2x80x94,
14) N3,
15) xe2x80x94N(R10)2,
16) R11OC(O)NR10xe2x80x94, or
17) C1-C6 alkyl, said alkyl optionally substituted with one or more of the following:
a) perfluoroalkyl,
b) halogen,
c) R10xe2x80x94,
d) R11S(O)mxe2x80x94,
e) R10C(O)NR10xe2x80x94,
f) (R10)2NC(O)xe2x80x94,
g) R102Nxe2x80x94C(NR10)xe2x80x94,
h) CN,
i) R10C(O)xe2x80x94,
j) R10OC(O)xe2x80x94,
k) N3,
l) xe2x80x94N(R10)2, or
m) R11OC(O)NR10xe2x80x94;
R10 is selected from:
1) hydrogen,
2) C1-C6 alkyl,
3) benzyl,
4) aryl, and
5) heterocyclyl;
R11 is selected from:
1) C1-C6 alkyl,
2) aryl, and
3) heterocyclyl;
A1 is selected from:
1) a bond,
2) xe2x80x94C(O)xe2x80x94,
3) xe2x80x94C(O)NR10xe2x80x94,
4) xe2x80x94NR10C(O)xe2x80x94,
5) O,
6) xe2x80x94N(R10)xe2x80x94,
7) xe2x80x94S(O)2N(R10)xe2x80x94,
8) xe2x80x94N(R10)S(O)2xe2x80x94, and
9) S(O)m;
A2 is selected from:
1) a bond,
2) xe2x80x94C(O)xe2x80x94,
3) xe2x80x94C(O)NR10xe2x80x94,
4) xe2x80x94NR10C(O)xe2x80x94,
5) O,
6) xe2x80x94N(R10)xe2x80x94,
7) xe2x80x94S(O)2N(R10)xe2x80x94,
8) xe2x80x94N(R10)S(O)2xe2x80x94,
9) S(O)m and
10) xe2x80x94C(R1d)2xe2x80x94;
W is heterocyclyl;
V is selected from: heterocyclyl and aryl;
X is selected from: a bond, xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94(CR1b2)nxe2x80x94 and xe2x80x94S(xe2x95x90O)mxe2x80x94;
Y is selected from: a bond, xe2x80x94C(xe2x95x90O)xe2x80x94 and xe2x80x94S(xe2x95x90O)mxe2x80x94;
Z1 is selected from:
1) aryl, and
2) heterocyclyl,
xe2x80x83said aryl and heterocyclyl optionally substituted with one or more R3, where R3 is:
a) C1-4 alkyl, unsubstituted or substituted with:
aa) C1-4 alkoxy,
bb) NR6R7,
cc) C3-6 cycloalkyl,
dd) aryl,
ee) heterocyclyl,
ff) OH,
gg) xe2x80x94S(O)mR4, or
hh) xe2x80x94C(O)NR6R7,
b) aryl,
c) heterocyclyl,
d) halogen,
e) OR6,
f) NR6R7,
g) CN,
h) NO2,
i) CF3,
j) xe2x80x94S(O)mR4,
k) xe2x80x94C(O)NR6R7, or
l) C3-C6 cycloalkyl;
Z2 is selected from:
1) a bond,
2) aryl, and
3) heterocyclyl,
xe2x80x83said aryl and heterocyclyl optionally substituted with one or more of the following:
a) C1-4 alkyl, optionally substituted with:
aa) C1-4 alkoxy,
bb) NR6R7,
cc) C3-6 cycloalkyl,
dd) aryl,
ee) heterocyclyl,
ff) OH,
gg) xe2x80x94S(O)mR4, or
hh) xe2x80x94C(O)NR6R7,
b) aryl,
c) heterocyclyl,
d) halogen,
e) OR6,
f) NR6R7,
g) CN,
h) NO2,
i) CF3,
j) xe2x80x94S(O)mR4,
k) xe2x80x94C(O)NR6R7, or
l) C3-C6 cycloalkyl;
or a pharmaceutically acceptable salt or stereoisomer thereof.
A second embodiment of this invention is the compound of formula A above, wherein:
R1a and R1d are independently selected from hydrogen and C1-C6 alkyl;
R1b and R1c are independently selected from:
1) hydrogen,
2) aryl,
3) heterocyclyl,
4) cycloalkyl,
5) R10Oxe2x80x94,
6) xe2x80x94N(R10)2,
7) C2-C6 alkenyl, and
8) C1-C6 alkyl, said alkyl optionally substituted with aryl, heterocyclyl, cycloalkyl, alkenyl, R10Oxe2x80x94 or N(R10)2;
R2 is selected from oxo, xe2x80x94(CO)NR6R7, and C1-6 alkyl, said alkyl optionally substituted with one or more of the following:
a) aryl,
b) heterocyclyl,
c) OR6,
d) SR4,
e) SO2R4, or
f) xe2x80x94(CO)NR6R7;
R4 is selected from C1-4 alkyl and C3-6 cycloalkyl,
xe2x80x83said alkyl and cycloalkyl optionally substituted with:
a) C1-4 alkoxy,
b) halogen,
c) aryl, or
d) heterocyclyl;
R6 and R7 are independently selected from:
1) H,
2) C1-6 alkyl,
3) C3-6 cycloalkyl,
4) R10C(O)xe2x80x94,
5) R10OC(O)xe2x80x94,
6) benzyl,
7) aryl, and
8) heterocyclyl,
xe2x80x83said alkyl, cycloalkyl, aryl and heterocyclyl optionally substituted with:
a) C1-4 alkoxy,
b) halogen,
c) aryl, or
d) heterocyclyl;
R8 is selected from:
1) aryl,
2) heterocyclyl,
3) C2-C6 alkenyl,
4) C2-C6 alkynyl,
5) C1-C6 perfluoroalkyl,
6) halogen,
7) R10Oxe2x80x94,
8) R10C(O)NR10xe2x80x94,
9) CN,
10) NO2,
11) (R10)2Nxe2x80x94C(NR10)xe2x80x94,
12) R10C(O)xe2x80x94,
13) N(R10)2,
14) R11OC(O)NR10xe2x80x94, and
15) C1-C6 alkyl, optionally substituted with:
a) aryl,
b) heterocyclyl,
c) C1-C6 perfluoroalkyl,
d) R10Oxe2x80x94,
e) R10C(O)NR10xe2x80x94,
f) (R10)2Nxe2x80x94C(NR10)xe2x80x94,
g) R10C(O)xe2x80x94,
h) xe2x80x94N(R10)2, or
i) R11OC(O)NR10xe2x80x94;
R9 is selected from:
1) C2-C6 alkenyl,
2) C2-C6 alkynyl,
3) C1-C6 perfluoroalkyl,
4) halogen,
5) R10Oxe2x80x94,
6) R11S(O)mxe2x80x94,
7) R10C(O)NR10xe2x80x94,
8) CN,
9) NO2,
10) (R10)2Nxe2x80x94C(NR10)xe2x80x94,
11) R10C(O)xe2x80x94,
12) xe2x80x94N(R10)2, or
13) R11OC(O)NR10xe2x80x94, and
14) C1-C6 alkyl, optionally substituted with one or more of the following:
a) C1-C6 perfluoroalkyl,
b) halogen,
c) R10Oxe2x80x94,
d) R11S(O)mxe2x80x94,
e) R10C(O)NR10xe2x80x94,
f) CN,
g) (R10)2Nxe2x80x94C(NR10)xe2x80x94,
h) R10C(O)xe2x80x94,
i) xe2x80x94N(R10)2, or
j) R11OC(O)NR10xe2x80x94;
R10 is selected from:
1) hydrogen,
2) C1-C6 alkyl,
3) benzyl,
4) aryl, and
5) heterocyclyl;
R11 is selected from:
1) C1-C6 alkyl,
2) aryl, and
3) heterocyclyl;
A1 is selected from:
1) a bond,
2) xe2x80x94C(O)xe2x80x94,
3) xe2x80x94C(O)NR10xe2x80x94,
4) xe2x80x94NR10C(O)xe2x80x94,
5) O,
6) xe2x80x94N(R10)xe2x80x94,
7) xe2x80x94S(O)2N(R10)xe2x80x94,
8) xe2x80x94N(R10)S(O)2xe2x80x94, and
9) S(O)m;
A2 is selected from:
1) a bond,
2) xe2x80x94C(O)xe2x80x94,
3) xe2x80x94C(O)NR10xe2x80x94,
4) xe2x80x94NR10C(O)xe2x80x94,
5) O,
6) xe2x80x94N(R10)xe2x80x94,
7) xe2x80x94S(O)2N(R10)xe2x80x94,
8) xe2x80x94N(R10)S(O)2xe2x80x94,
9) S(O)m, and
10) xe2x80x94C(R1d)2xe2x80x94;
V is selected from:
1) heterocyclyl, wherein heterocyclyl is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl, and
2) aryl, wherein aryl is phenyl or naphthyl;
W is a heterocyclyl selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, and isoquinolinyl;
X is a bond, xe2x80x94(CR1b2)nxe2x80x94 or xe2x80x94C(xe2x95x90O)xe2x80x94;
Y is a bond or xe2x80x94C(xe2x95x90O)xe2x80x94;
Z1 is selected from aryl and heterocyclyl,
xe2x80x83said aryl and heterocyclyl optionally substituted with one or two of R3, where R3 is:
a) C1-4 alkyl, optionally substituted with:
aa) C1-4 alkoxy,
bb) NR6R7,
cc) C3-6 cycloalkyl,
dd) aryl,
ee) heterocyclyl,
ff) OH,
gg) xe2x80x94S(O)mR4, or
hh) xe2x80x94C(O)NR6R7,
b) aryl,
c) heterocyclyl,
d) halogen,
e) OR6,
f) NR6R7,
g) CN,
h) NO2,
i) CF3,
j) xe2x80x94S(O)mR4,
k) xe2x80x94C(O)NR6R7, or
1) C3-C6 cycloalkyl;
Z2 is selected from a bond, aryl, and heterocyclyl,
xe2x80x83said aryl and heterocyclyl optionally substituted with one or two of:
a) C1-4 alkyl, optionally substituted with:
aa) C1-4 alkoxy,
bb) NR6R7,
cc) C3-6 cycloalkyl,
dd) aryl,
ee) heterocyclyl,
ff) OH,
gg) xe2x80x94S(O)mR4, or
hh) xe2x80x94C(O)NR6R7,
b) aryl,
c) heterocyclyl,
d) halogen,
e) OR6,
f) NR6R7,
g) CN,
h) NO2,
i) CF3,
j) xe2x80x94S(O)mR4,
k) xe2x80x94C(O)NR6R7, or
l) C3-C6 cycloalkyl;
or a pharmaceutically acceptable salt or stereoisomer thereof.
A third embodiment of this invention is a compound of Formula A as described immediately above, wherein:
R10 is selected from hydrogen, C1-C6 alkyl, benzyl and aryl;
R11 is selected from C1-C6 alkyl and aryl;
A1 is selected from: a bond, xe2x80x94C(O)xe2x80x94 and O;
or a pharmaceutically acceptable salt or stereoisomer thereof.
Yet another embodiment of the invention is the compound of Formula A described above, wherein V is phenyl.
And still another embodiment of the invention is a compound of Formula B 
wherein:
X is a bond or xe2x80x94C(R1b)2xe2x80x94;
Y is a bond;
and all other variables are as defined above.
A further embodiment is a compound of Formula C 
wherein x is 0, 1, or 2;
Z1 is aryl or heterocyclyl;
R3 is:
1) C1-6 alkyl,
2) aryl,
3) heterocyclyl,
4) halogen,
5) OR6,
6) NR6R7,
7) CN,
8) NO2,
9) CF3,
10) xe2x80x94S(O)mR4,
11) xe2x80x94C(O)NR6R7, or
12) C3-C6 cycloalkyl;
and all other variables are as defined above.
And another embodiment is illustrated by the compounds of Formula D 
wherein,
Z1 is phenyl or naphthyl;
V is phenyl or naphthyl;
and all other variables are as defined above.
The preferred compounds of this invention are as follows:
(5S,22S)-19,20-dihydro-3-methyl-19-oxo-5H-5,22:18,21-diethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriazacyclooctadecine-9-carbonitrile;
(5R,22R)-19,20-dihydro-3-methyl-19-oxo-5H-5,22:18,21-diethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriazacyclooctadecine-9-carbonitrile;
(5S,22R)-19,20-dihydro-3-methyl-19-oxo-5H-5,22:18,21-diethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriazacyclooctadecine-9-carbonitrile;
(5R,22S)-19,20-dihydro-3-methyl-19-oxo-5H-5,22:18,21-diethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriazacyclooctadecine-9-carbonitrile;
(xc2x1)-(5R*,22R*)-19,20-dihydro-3-methyl-19-oxo-5H-5,22:18,21-diethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriazacyclooctadecine-9-carbonitrile; and
(xc2x1)-(5R*,22S*)-19,20-dihydro-3-methyl-19-oxo-5H-5,22:18,21-diethano-12,14-etheno-6,10-metheno-22H-benzo[d]imidazo[4,3-k][1,6,9,12]oxatriazacyclooctadecine-9-carbonitrile, or a pharmaceutically acceptable salt thereof.
The compounds of the present invention may have asymmetric centers, chiral axes, and chiral planes (as described in: E. L. Eliel and S. H. Wilen, Stereochemistry of Carbon Compounds, John Wiley and Sons, New York, 1994, pages 1119-1190), and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention. When any variable (e.g. aryl, heterocyclyl, R1, R2 etc.) occurs more than one time in any constituent, its definition on each occurence is independent at every other occurence. Also, combinations of substituents/or variables are permissible only if such combinations result in stable compounds.
As used herein, xe2x80x9calkylxe2x80x9d is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms; xe2x80x9calkoxyxe2x80x9d represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge. xe2x80x9cHalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as used herein means fluoro, chloro, bromo and iodo.
As used herein, xe2x80x9carylxe2x80x9d is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl.
The term xe2x80x9cheterocyclylxe2x80x9d or xe2x80x9cheterocyclicxe2x80x9d, as used herein, represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic heterocyclic ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. The term heterocyclyl or heterocyclic includes heteroaryl moieties. Examples of such heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, 2-oxopiperazinyl, 2-oxopiperdinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl.
As used herein, xe2x80x9cheteroarylxe2x80x9d is intended to mean any stable monocyclic or bicyclic carbon ring of up to 7 members in each ring, wherein at least one ring is aromatic and wherein from one to four carbon atoms are replaced by heteroatoms selected from the group consisting of N, O, and S. Examples of such heteroaryl elements include, but are not limited to, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl, dihydrobenzothiopyranyl, dihydrobenzothiopyranyl sulfone, furyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxadiazolyl, pyridyl, pyrazinyl, pyrazolyl, pyridazinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, thiazolyl, thienofuryl, thienothienyl, and thienyl.
When t is at least 2, two R2""s can be on the same carbon and can be combined to form xe2x80x94(CH2)uxe2x80x94, such that a cyclic moiety is formed. Examples of such cyclic moieties include, but are not limited to: 
In addition, such cyclic moieties may optionally include a heteroatom(s). Examples of such heteroatom-containing cyclic moieties include, but are not limited to: 
Lines drawn into the ring systems from substituents (such as from R2, R3, R4 etc.) indicate that the indicated bond may be attached to any of the substitutable ring carbon atoms.
When they are attached to the same atom, R6 and R7 or R5 and R7 may be joined to form a heterocyclic ring, which includes the heteroatom to which R5, R6 and R7 is attached. Examples of ring systems that are formed when R6 and R7 or R5 and R7 are joined are shown below: 
This list is illustrative and not exhaustive. Other possibilities would be readily apparent to the skilled artisan.
Preferably, R1a and R1b are independently selected from: hydrogen, xe2x80x94N(R10)2, R10C(O)NR10xe2x80x94 or unsubstituted or substituted C1-C6 alkyl wherein the substituent on the substituted C1-C6 alkyl is selected from unsubstituted or substituted phenyl, xe2x80x94N(R10)2, R10Oxe2x80x94 and R10C(O)NR10xe2x80x94. Most preferably R1a and R1b are H.
Preferably, R1c is selected from: hydrogen, or unsubstituted or substituted C1-C6 alkyl wherein the substituent on the substituted C1-C6 alkyl is selected from unsubstituted or substituted phenyl, xe2x80x94N(R10)2, R10Oxe2x80x94 and R10C(O)NR10xe2x80x94. Most preferably R1c is H.
Preferably R2 is oxo or C1-C8 alkyl.
Preferably, R4, R5, R6, and R7 are selected from: hydrogen, C1-C6 alkyl, and aryl.
Preferably, R8 is CN, aryl, heterocyclyl or C1-C6 alkyl.
Preferably, R9 is hydrogen or C1-C6 alkyl.
Preferably, R10 is selected from H, C1-C6 alkyl and benzyl.
Preferably, A1 and A2 are independently selected from: a bond, xe2x80x94C(O)NR10xe2x80x94, xe2x80x94NR10C(O)xe2x80x94, O, xe2x80x94N(R10)xe2x80x94, xe2x80x94S(O)mxe2x80x94, xe2x80x94S(O)2N(R10)xe2x80x94 and xe2x80x94N(R10)S(O)2xe2x80x94, wherein m is 0, 1 or 2.
Preferably, V is selected from heteroaryl and aryl.
More preferably, V is phenyl.
Preferably, X and Y are independently selected from: a bond and xe2x80x94C(xe2x95x90O)xe2x80x94.
More preferably, X and Y are a bond.
Preferably, Z1 and Z2 are independently selected from unsubstituted or substituted phenyl, unsubstituted or substituted naphthyl, unsubstituted or substituted pyridyl, unsubstituted or substituted furanyl and unsubstituted or substituted thienyl.
More preferably, Z1 is selected from unsubstituted or substituted phenyl and unsubstituted or substituted naphthyl.
More preferably, Z2 is selected from a bond and unsubstituted or substituted phenyl.
Preferably, W is selected from imidazolinyl, imidazolyl, oxazolyl, pyrazolyl, pyyrolidinyl, thiazolyl and pyridyl. More preferably, W is selected from imidazolyl and pyridyl.
Preferably, n is 0, 1, or 2. Most preferably n is 0. Preferably, r is 1 or 2. Preferably p is 2. Preferably s is 0 or 1. Most preferably s is 0. Preferably, the moiety 
is selected from: 
It is intended that the definition of any substituent or variable (e.g., R1a, R9, n, etc.) at a particular location in a molecule be independent of its definitions elsewhere in that molecule. Thus, xe2x80x94N(R10)2 represents xe2x80x94NH2, xe2x80x94NHCH3, xe2x80x94NHC2H5, etc. It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials.
The pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed, e.g., from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.
The pharmaceutically acceptable salts of the compounds of this invention can be synthesized from the compounds of this invention which contain a basic moiety by conventional chemical methods. Generally, the salts are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.
Reactions used to generate the compounds of this invention are prepared by employing reactions as shown in the Schemes below, in addition to other standard manipulations such as ester hydrolysis, cleavage of protecting groups, etc., as may be known in the literature or exemplified in the experimental procedures. Substituents R, Ra, Rb and Rsub, as shown in the Schemes, represent the substituents R2, R3, R4, and R5, and substituents on Z1 and Z2; however their point of attachment to the ring is illustrative only and is not meant to be limiting.
These reactions may be employed in a linear sequence to provide the compounds of the invention or they may be used to synthesize fragments which are subsequently joined by the reactions described in the schemes.
Synopsis of the Schemes:
The requisite intermediates are in some cases commercially available, or can be prepared according to literature procedures. In Scheme 1, for example, the synthesis of the piperazinone portion of the molecule is outlined. Preparation of the substituted piperazine intermediates is essentially that described by J. S. Kiely and S. R. Priebe in Organic Preparations and Proceedings Int., 1990, 22, 761-768.
Scheme 2 illustrates one possible route to the imidazolyl portion of the molecule. As shown in Scheme 3, an aldehyde and an amine are condensed to form a new ring. Following alcohol deprotection, reaction of the intermediate with cesium carbonate results in nucleophilic aromatic substitution reaction to yield a compound of the instant invention. This cyclization reaction depends on the presence of an electron withdrawing moiety (such as nitro, cyano, and the like) either ortho or para to the fluorine atom. When an ortho or para electron withdrawing group is not present on the electrophilic intermediate, the intramolecular cyclization may be accomplished via an Ullman reaction or other protocol that would be apparent to those skilled in the art.
Schemes 4 and 5 illustrate one possible route to the synthesis of the compounds wherein heterocycles other than imidazole comprise the W substituent. Scheme 6 presents one possible approach to the synthesis of variably substituted piperazinediones and piperazines from suitably protected amino acids. Schemes 7 and 8 show protocols for the preparation of variably substituted piperazine intermediates that can be incorporated into the cylization schemes described above to arrive at further compounds of the instant invention. For example, incorporation of a spirocyclic. moiety (for example, when two R2""s are combined to form a ring) is illustrated in Scheme 8. 
(a) ClCH2COCl, aq NaHCO3, i-PrOAc, 0xc2x0 C.; ethanolamine, 55xc2x0 C., 1 h.
(b) Boc2O, THF, 0xc2x0 C.
(c) di-tert-butylazodicarboxylate, Bu3P, THF, 0xc2x0 C.-RT
(d) BnBr, K2CO3, acetone, reflux.
(e) HCl, EtOAc, 0xc2x0 C.; NaHCO3: 
(f) NBS, AIBN, CCl4, reflux.
(g) AgNO3, EtOH, reflux.
(h) allyl-MgBr, THF, xe2x88x9278xc2x0 C.
(i) Zn(CN)2 Pd(PPh3)4, DMF, 80xc2x0 C.
(j) Ms2O, Et3N, CH2Cl2, 0xc2x0 C.
(k) 2-Me-imidazole, K2CO3, DMF, 100xc2x0 C.
(l) 9-BBN, THF, 0xc2x0 C.-rt; H2O2, NaHCO3xe2x80x94H2O.
(m) (COCl)2, DMSO, Et3N, CH2Cl2, xe2x88x9278xc2x0 C.-rt. 
(n) MgSO4, PhCl, reflux.
(o) Pd/C, H2, MeOH-EtOAc.
(p) Cs2CO3, DMSO, 80xc2x0 C. 
In a preferred embodiment of the instant invention the compounds of the invention are selective inhibitors of farnesyl-protein transferase. A compound is considered a selective inhibitor of farnesyl-protein transferase, for example, when its in vitro farnesyl-protein transferase inhibitory activity, as assessed by the assay described in Example 7, is at least 100 times greater than the in vitro activity of the same compound against geranylgeranyl-protein transferase-type I in the assay described in Example 8. Preferably, a selective compound exhibits at least 1000 times greater activity against one of the enzymatic activities when comparing geranylgeranyl-protein transferase-type I inhibition and farnesyl-protein transferase. inhibition.
It is also preferred that the selective inhibitor of farnesyl-protein transferase is further characterized by:
a) an IC50 (a measure of in vitro inhibitory activity) for inhibition of the prenylation of newly synthesized K-Ras protein more than about 100-fold higher than the IC50 for the inhibition of the farnesylation of hDJ protein.
When measuring such IC50s the assays described in Examples 12 and 13 may be utilized.
It is also preferred that the selective inhibitor of farnesyl-protein transferase is further characterized by:
b) an IC50 (a measurement of in vitro inhibitory activity) for inhibition of K4B-Ras dependent activation of MAP kinases in cells at least 100-fold greater than the IC50 for inhibition of the farnesylation of the protein hDJ in cells.
It is also preferred that the selective inhibitor of farnesyl-protein transferase is further characterized by:
c) an IC50 (a measurement of in vitro inhibitory activity) against H-Ras dependent activation of MAP kinases in cells at least 1000 fold lower than the inhibitory activity (IC50) against H-ras-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells.
When measuring Ras dependent activation of MAP kinases in cells the assays described in Example 11 may be utilized.
In another preferred embodiment of the instant invention the compounds of the invention are dual inhibitors of farnesyl-protein transferase and geranylgeranyl-protein transferase type I. Such a dual inhibitor may be termed a Class II prenyl-protein transferase inhibitor and will exhibit certain characteristics when assessed in in vitro assays, which are dependent on the type of assay employed.
In a SEAP assay, such as described in Examples 11, it is preferred that the dual inhibitor compound has an in vitro inhibitory activity (IC50) that is less than about 12 xcexcM against K4B-Ras dependent activation of MAP kinases in cells.
The Class II prenyl-protein transferase inhibitor may also be characterized by:
a) an IC50 (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells between 0.1 and 100 times the IC50 for inhibiting the farnesylation of the protein hDJ in cells; and
b) an IC50 (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells greater than 5-fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.
The Class II prenyl-protein transferase inhibitor may also be characterized by:
a) an IC50 (a measurement of in vitro inhibitory activity) against H-Ras dependent activation of MAP kinases in cells greater than 2 fold lower but less than 20,000 fold lower than the inhibitory activity (IC50) against H-ras-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and
b) an IC50 (a measurement of in vitro inhibitory activity) against H-ras-CVLL dependent activation of MAP kinases in cells greater than 5-fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.
The Class II prenyl-protein transferase inhibitor may also be characterized by:
a) an IC50 (a measurement of in vitro inhibitory activity) against H-Ras dependent activation of MAP kinases in cells greater than 10-fold lower but less than 2,500 fold lower than the inhibitory activity (IC50) against H-ras-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and
b) an IC50 (a measurement of in vitro inhibitory activity) against H-ras-CVLL dependent activation of MAP kinases in cells greater than 5 fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.
A method for measuring the activity of the inhibitors of prenyl-protein transferase, as well as the instant combination compositions, utilized in the instant methods against Ras dependent activation of MAP kinases in cells is described in Example 11.
The instant compounds are useful as pharmaceutical agents for mammals, especially for humans. These compounds may be administered to patients for use in the treatment of cancer. Examples of the type of cancer which may be treated with the compounds of this invention include, but are not limited to, colorectal carcinoma, exocrine pancreatic carcinoma, myeloid leukemias and neurological tumors. Such tumors may arise by mutations in the ras genes themselves, mutations in the proteins that can regulate Ras activity (i.e., neurofibromin (NF-1), neu, src, abl, lck, fyn) or by other mechanisms.
The compounds of the instant invention inhibit farnesyl-protein transferase and the farnesylation of the oncogene protein Ras. The instant compounds may also inhibit tumor angiogenesis, thereby affecting the growth of tumors (J. Rak et al. Cancer Research, 55:4575-4580 (1995)). Such anti-angiogenesis properties of the instant compounds may also be useful in the treatment of certain forms of vision deficit related to retinal vascularization.
The compounds of this invention are also useful for inhibiting other proliferative diseases, both benign and malignant, wherein Ras proteins are aberrantly activated as a result of oncogenic mutation in other genes (i.e., the Ras gene itself is not activated by mutation to an oncogenic form) with said inhibition being accomplished by the administration of an effective amount of the compounds of the invention to a mammal in need of such treatment. For example, the composition is useful in the treatment of neurofibromatosis, which is a benign proliferative disorder.
The instant compounds may also be useful in the treatment of certain viral infections, in particular in the treatment of hepatitis delta and related viruses (J. S. Glenn et al. Science, 256:1331-1333 (1992).
The compounds of the instant invention are also useful in the prevention of restenosis after percutaneous transluminal coronary angioplasty by inhibiting neointimal formation (C. Indolfi et al. Nature medicine, 1:541-545(1995).
The instant compounds may also be useful in the treatment and prevention of polycystic kidney disease (D. L. Schaffner et al. American Journal of Pathology, 142:1051-1060 (1993) and B. Cowley, Jr. et al. FASEB Journal, 2:A3160 (1988)).
The instant compounds may also be useful for the treatment of fungal infections.
The instant compounds may also be useful as inhibitors of proliferation of vascular smooth muscle cells and therefore useful in the prevention and therapy of arteriosclerosis and diabetic vascular pathologies.
The compounds of the instant invention may also be useful in the prevention and treatment of endometriosis, uterine fibroids, dysfunctional uterine bleeding and endometrial hyperplasia.
In such methods of prevention and treatment as described herein, the prenyl-protein transferase inhibitors of the instant invention may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated. For example, the prenyl-protein transferase inhibitor may be useful in further combination with drugs known to supress the activity of the ovaries and slow the growth of the endometrial tissue. Such drugs include but are not limited to oral contraceptives, progestins, danazol and GnRH (gonadotropin-releasing hormone) agonists.
Administration of the prenyl-protein transferase inhibitor may also be combined with surgical treatment of endometriosis (such as surgical removal of misplaced endometrial tissue) where appropriate.
The instant compounds may also be useful as inhibitors of corneal inflammation. These compounds may improve the treatment of corneal opacity which results from cauterization-induced corneal inflammation. The instant compounds may also be useful in reducing corneal edema and neovascularization. (K. Sonoda et al., Invest. Ophthalmol. Vis. Sci., 1998, vol. 39, p 2245-2251).
The compounds of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The compounds can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.
Additionally, the compounds of the instant invention may be administered to a mammal in need thereof using a gel extrusion mechanism (GEM) device, such as that described in U.S. Ser. No. 60/144,643, filed on Jul. 20, 1999, which is hereby incorporated by reference.
As used herein, the term xe2x80x9ccompositionxe2x80x9d is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.
The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropyl-methylcellulose or hydroxypropyl-cellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring agents, preservatives and antioxidants.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer""s solution and isotonic sodium chloride solution.
The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.
The injectable solutions or microemulsions may be introduced into a patient""s blood-stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized. An example of such a device is the Deltec CADD-PLUS(trademark) model 5400 intravenous pump.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
Compounds of Formula A may also be administered in the form of a suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compound of Formula A are employed. (For purposes of this application, topical application shall include mouth washes and gargles.)
The compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. Compounds of the present invention may also be delivered as a suppository employing bases such as cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
When a compound according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, sex and response of the individual patient, as well as the severity of the patient""s symptoms.
In one exemplary application, a suitable amount of compound is administered to a mammal undergoing treatment for cancer. Administration occurs in an amount between about 0.1 mg/kg of body weight to about 60 mg/kg of body weight per day, preferably of between 0.5 mg/kg of body weight to about 40 mg/kg of body weight per day.
The compounds of the instant invention may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated. For example, the compounds of the instant invention may also be co-administered with other well known cancer therapeutic agents that are selected for their particular usefulness against the condition that is being treated. Included in such combinations of therapeutic agents are combinations of the instant prenyl-protein transferase inhibitors and an antineoplastic agent. It is also understood that such a combination of antineoplastic agent and inhibitor of prenyl-protein transferase may be used in conjunction with other methods of treating cancer and/or tumors, including radiation therapy and surgery. It is further understood that any of the therapeutic agents described herein may also be used in combination with a compound of the instant invention and an antineoplastic agent.
Examples of an antineoplastic agent include, in general, microtubule-stabilizing agents such as paclitaxel (also known as Taxol(copyright)), docetaxel (also known as Taxotere(copyright)), epothilone A, epothilone B, desoxyepothilone A, desoxyepothilone B or their derivatives); microtubule-disruptor agents; alkylating agents, for example, nitrogen mustards, ethyleneimine compounds, alkyl sulfonates and other compounds with an alkylating action such as nitrosoureas, cisplatin, and dacarbazine; anti-metabolites, for example, folic acid, purine or pyrimidine antagonists; epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes; biological response modifiers and growth inhibitors; mitotic inhibitors, for example, vinca alkaloids and derivatives of podophyllotoxin; cytotoxic antibiotics; hormonal/anti-hormonal therapeutic agents, haematopoietic growth factors and antibodies (such as trastuzumab, also known as Herceptin(trademark)).
Example classes of antineoplastic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the taxanes, the epothilones, discodermolide, the pteridine family of drugs, diynenes and the podophyllotoxins. Particularly useful members of those classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro-methotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin or podo-phyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine, paclitaxel and the like. Other useful antineoplastic agents include estramustine, cisplatin, carboplatin, cyclophosphamide, bleomycin, tamoxifen, ifosamide, melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase, dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), procarbazine, mitomycin, cytarabine, etoposide, methotrexate, bleomycin, chlorambucil, camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives, interferons and interleukins. Particular examples of antineoplastic, or chemotherapeutic, agents are described, for example, by D. J. Stewart in xe2x80x9cNausea and Vomiting: Recent Research and Clinical Advancesxe2x80x9d, Eds. J. Kucharczyk, et al., CRC Press Inc., Boca Raton, Fla., USA (1991), pages 177-203, especially page 188. See also, R. J. Gralla, et al., Cancer Treatment Reports, 68(1), 163-172 (1984).
The preferred class of antineoplastic agents is the taxanes and the preferred antineoplastic agent is paclitaxel.
The compounds of the instant invention may also be co-administered with antisense oligonucleotides which are specifically hybridizable with RNA or DNA deriving from human ras gene. Such antisense oligonucleotides are described in U.S. Pat. No. 5,576,208 and PCT Publication No. WO 99/22772. The instant compounds are particularly useful when co-administered with the antisense oligonucleotide comprising the amino acid sequence of SEQ.ID.NO: 2 of U.S. Pat. No. 5,576,208.
Certain compounds of the instant invention may exhibit very low plasma concentrations and significant inter-individual variation in the plasma levels of the compound. It is believed that very low plasma concentrations and high intersubject variability achieved following administration of certain prenyl-protein transferase inhibitors to mammals may be due to extensive metabolism by cytochrome P450 enzymes prior to entry of drug into the systemic circulation. Prenyl-protein transferase inhibitors may be metabolized by cytochrome P450 enzyme systems, such as CYP3A4, CYP2D6, CYP2C9, CYP2C19 or other cytochrome P450 isoform. If a compound of the instant invention demonstrates an affinity for one or more of the cytochrome P450 enzyme systems, another compound with a higher affinity for the P450 enzyme(s) involved in metabolism should be administered concomitantly. Examples of compounds that have a comparatively very high affinity for CYP3A4, CYP2D6, CYP2C9, CYP2C19 or other P450 isoform include, but are not limited to, piperonyl butoxide, troleandomycin, erythromycin, proadifen, isoniazid, allyliso-propylacetamide, ethinylestradiol, chloramphenicol, 2-ethynylnaphthalene and the like. Such a high affinity compound, when employed in combination with a compound of formula A, may reduce the inter-individual variation and increase the plasma concentration of a compound of formula A to a level having substantial therapeutic activity by inhibiting the metabolism of the compound of formula A. Additionally, inhibiting the metabolism of a compound of the instant invention prolongs the pharmacokinetic half-life, and thus the pharmacodynamic effect, of the compound.
A compound of the present invention may be employed in conjunction with antiemetic agents to treat nausea or emesis, including acute, delayed, late-phase, and anticipatory emesis, which may result from the use of a compound of the present invention, alone or with radiation therapy. For the prevention or treatment of emesis a compound of the present invention may be used in conjunction with other anti-emetic agents, especially neurokinin-1 receptor antagonists, 5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and zatisetron, GABAB receptor agonists, such as baclofen, or a corticosteroid such as Decadron (dexamethasone), Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as disclosed in U.S. Pat. Nos. 2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326 and 3,749,712. For the treatment or prevention of emesis, conjunctive therapy with a neurokinin-1 receptor antagonist, a 5HT3 receptor antagonist and a corticosteroid is preferred.
Neurokinin-1 receptor antagonists of use in conjunction with the compounds of the present invention are fully described, for example, in U.S. Pat. Nos. 5,162,339, 5,232,929, 5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699, 5,719,147; European Patent Publication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429 366, 0 430 771, 0 436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902, 0 514 273, 0 514 274, 0 514 275, 0 514 276, 0 515 681, 0 517 589, 0 520 555, 0 522 808, 0 528 495, 0 532 456, 0 533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0 599 538, 0 610 793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707 006, 0 708 101, 0 709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT International Patent Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688, 92/06079, 92/12151, 92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330, 93/00331, 93/01159, 93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084, 93/14113, 93/18023, 93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402, 94/02461, 94/02595, 94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997, 94/10165, 94/10167, 94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903, 94/19320, 94/19323, 94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042, 95/06645, 95/07886, 95/07908, 95/08549, 95/11880, 95/14017, 95/15311, 95/16679, 95/17382, 95/18124, 95/18129, 95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418, 95/30674, 95/30687, 95/33744, 96/05181, 96/05193, 96/05203, 96/06094, 96/07649, 96/10562, 96/16939, 96/18643, 96/20197, 96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214, 96/32385, 96/37489, 97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206, 97/19084, 97/19942 and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2 269 170, 2 269 590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689. The preparation of such compounds is fully described in the aforementioned patents and publications.
A particularly preferred neurokinin-1 receptor antagonist for use in conjunction with the compounds of the present invention is 2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluorophenyl)-4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine, or a pharmaceutically acceptable salt thereof, which is described in U.S. Pat. No. 5,719,147.
For the treatment of cancer, it may be desirable to employ a compound of the present invention in conjunction with another pharmacologically active agent(s). A compound of the present invention and the other pharmacologically active agent(s) may be administered to a patient simultaneously, sequentially or in combination. For example, the present compound may employed directly in combination with the other active agent(s), or it may be administered prior, concurrent or subsequent to the administration of the other active agent(s). In general, the currently available dosage forms of the known therapeutic agents for use in such combinations will be suitable.
For example, a compound of the present invention may be presented together with another therapeutic agent in a combined preparation, such as with an antiemetic agent for simultaneous, separate, or sequential use in the relief of emesis associated with employing a compound of the present invention and radiation therapy. Such combined preparations may be, for example, in the form of a twin pack. A preferred combination comprises a compound of the present invention with antiemetic agents, as described above.
Radiation therapy, including x-rays or gamma rays which are delivered from either an externally applied beam or by implantation of tiny radioactive sources, may also be used in combination with the instant inhibitor of prenyl-protein transferase alone to treat cancer.
Additionally, compounds of the instant invention may also be useful as radiation sensitizers, as described in WO 97/38697, published on Oct. 23, 1997, and herein incorporated by reference.
The instant compounds may also be useful in combination with other inhibitors of parts of the signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation. Thus, the instant compounds may be utilized in combination with farnesyl pyrophosphate competitive inhibitors of the activity of farnesyl-protein transferase or in combination with a compound which has Raf antagonist activity. The instant compounds may also be co-administered with compounds that are selective inhibitors of geranylgeranyl protein transferase.
In particular, if the compound of the instant invention is a selective inhibitor of farnesyl-protein transferase, co-administration with a compound(s) that is a selective inhibitor of geranylgeranyl protein transferase may provide an improved therapeutic effect.
In particular, the compounds disclosed in the following patents and publications may be useful as farnesyl pyrophosphate-competitive inhibitor component of the instant composition: U.S. Ser. Nos. 08/254,228 and 08/435,047. Those patents and publications are incorporated herein by reference.
In practicing methods of this invention, which comprise administering, simultaneously or sequentially or in any order, two or more of a protein substrate-competitive inhibitor and a farnesyl pyrophosphate-competitive inhibitor, such administration can be orally or parenterally, including intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration. It is preferred that such administration be orally. It is more preferred that such administration be orally and simultaneously. When the protein substrate-competitive inhibitor and farnesyl pyrophosphate-competitive inhibitor are administered sequentially, the administration of each can be by the same method or by different methods.
The instant compounds may also be useful in combination with an integrin antagonist for the treatment of cancer, as described in U.S. Ser. No. 09/055,487, filed Apr. 6, 1998, and WO 98/44797, published on Oct. 15, 1998, which are incorporated herein by reference.
As used herein the term an integrin antagonist refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to an integrin(s) that is involved in the regulation of angiogenisis, or in the growth and invasiveness of tumor cells. In particular, the term refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the xcex1vxcex23 integrin, which selectively antagonize, inhibit or counteract binding of a physiological ligand to the xcex1vxcex25 integrin, which antagonize, inhibit or counteract binding of a physiological ligand to both the xcex1vxcex23 integrin and the xcex1vxcex25 integrin, or which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the xcex11xcex21, xcex12xcex21, xcex15xcex21, xcex16xcex21 and xcex16xcex24 integrins. The term also refers to antagonists of any combination of xcex1vxcex23 integrin, xcex1vxcex25 integrin, xcex11xcex21, xcex12xcex21, xcex15xcex21, xcex16xcex21 and xcex16xcex24 integrins. The instant compounds may also be useful with other agents that inhibit angiogenisis and thereby inhibit the growth and invasiveness of tumor cells, including, but not limited to angiostatin and endostatin.
The instant compounds may also be useful in combination with an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) for the treatment of cancer. Compounds which have inhibitory activity for HMG-CoA reductase can be readily identified by using assays well-known in the art. For example, see the assays described or cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131 at pp. 30-33. The terms xe2x80x9cHMG-CoA reductase inhibitorxe2x80x9d and xe2x80x9cinhibitor of HMG-CoA reductasexe2x80x9d have the same meaning when used herein.
Examples of HMG-CoA reductase inhibitors that may be used include but are not limited to lovastatin (MEVACOR(copyright); see U.S. Pat. Nos. 4,231,938; 4,294,926; 4,319,039), simvastatin (ZOCOR(copyright); see U.S. Pat. Nos. 4,444,784; 4,820,850; 4,916,239), pravastatin (PRAVACHOL(copyright); see U.S. Pat. Nos. 4,346,227; 4,537,859; 4,410,629; 5,030,447 and 5,180,589), fluvastatin (LESCOL(copyright)); see U.S. Pat. Nos. 5,354,772; 4,911,165; 4,929,437; 5,189,164; 5,118,853; 5,290,946; 5,356,896), atorvastatin (LIPITOR(copyright); see U.S. Pat. Nos. 5,273,995; 4,681,893; 5,489,691; 5,342,952) and cerivastatin (also known as rivastatin and BAYCHOL(copyright); see U.S. Pat. No. 5,177,080). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, xe2x80x9cCholesterol Lowering Drugsxe2x80x9d, Chemistry and Industry, pp. 85-89 (Feb. 5, 1996) and U.S. Pat. Nos. 4,782,084 and 4,885,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open-acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such salts, esters, open-acid and lactone forms is included within the scope of this invention. An illustration of the lactone portion and its corresponding open-acid form is shown below as structures I and II. 
In HMG-CoA reductase inhibitors where an open-acid form can exist, salt and ester forms may preferably be formed from the open-acid, and all such forms are included within the meaning of the term xe2x80x9cHMG-CoA reductase inhibitorxe2x80x9d as used herein. Preferably, the HMG-CoA reductase inhibitor is selected from lovastatin and simvastatin, and most preferably simvastatin. Herein, the term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d with respect to the HMG-CoA reductase inhibitor shall mean non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base, particularly those, formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium, as well as those salts formed from amines such as ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,Nxe2x80x2-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidine-1xe2x80x2-yl-methylbenzimidazole, diethylamine, piperazine, and tris(hydroxymethyl) aminomethane. Further examples of salt forms of HMG-CoA reductase inhibitors may include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate.
Ester derivatives of the described HMG-CoA reductase inhibitor compounds may act as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, may cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy.
Similarly, the instant compounds may be useful in combination with agents that are effective in the treatment and prevention of NF-1, restenosis, polycystic kidney disease, infections of hepatitis delta and related viruses and fungal infections.
If formulated as a fixed dose, such combination products employ the combinations of this invention within the dosage range described above and the other pharmaceutically active agent(s) within its approved dosage range. Combinations of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a multiple combination formulation is inappropriate.
The instant compounds may also be useful in combination with prodrugs of antineoplastic agents. In particular, the instant compounds may be co-administered either concurrently or sequentially with a conjugate (termed a xe2x80x9cPSA conjugatexe2x80x9d) which comprises an oligopeptide, that is selectively cleaved by enzymatically active prostate specific antigen (PSA), and an antineoplastic agent. Such co-administration will be particularly useful in the treatment of prostate cancer or other cancers which are characterized by the presence of enzymatically active PSA in the immediate surrounding cancer cells, which is secreted by the cancer cells.
Compounds which are PSA conjugates and are therefore useful in such a co-administration, and methods of synthesis thereof, can be found in the following patents, pending patent applications and publications which are herein incorporated by reference:
U.S. Pat. No. 5,599,686, granted on Feb. 4, 1997;
WO 96/00503 (Jan. 11, 1996); U.S. Ser. No. 08/404,833, filed on Mar. 15, 1995;
U.S. Ser. No. 08/468,161, filed on Jun. 6, 1995;
U.S. Pat. No. 5,866,679, granted on Feb. 2, 1999;
WO 98/10651 (Mar. 19, 1998); U.S. Ser. No. 08/926,412, filed on Sep. 9, 1997;
WO 98/18493 (May 7, 1998); U.S. Ser. No. 08/950,805, filed on Oct. 14, 1997;
WO 99/02175 (Jan. 21, 1999); U.S. Ser. No. 09/112,656, filed on Jul. 9, 1998; and
WO 99/28345 (Jun. 10, 1999); U.S. Ser. No. 09/193,365, filed on Nov. 17, 1998.
Compounds which are described as prodrugs wherein the active therapeutic agent is released by the action of enzymatically active PSA and therefore may be useful in such a co-administration, and methods of synthesis thereof, can be found in the following patents, pending patent applications and publications, which are herein incorporated by reference: WO 98/52966 (Nov. 26, 1998).
All patents, publications and pending patent applications identified are herein incorporated by reference.
The compounds of the instant invention are also useful as a component in an assay to rapidly determine the presence and quantity of farnesyl-protein transferase (FPTase) in a composition. Thus the composition to be tested may be divided and the two portions contacted with mixtures which comprise a known substrate of FPTase (for example a tetrapeptide having a cysteine at the amine terminus) and farnesyl pyrophosphate and, in one of the mixtures, a compound of the instant invention. After the assay mixtures are incubated for an sufficient period of time, well known in the art, to allow the FPTase to farnesylate the substrate, the chemical content of the assay mixtures may be determined by well known immunological, radiochemical or chromatographic techniques. Because the compounds of the instant invention are selective inhibitors of FPTase, absence or quantitative reduction of the amount of substrate in the assay mixture without the compound of the instant invention relative to the presence of the unchanged substrate in the assay containing the instant compound is indicative of the presence of FPTase in the composition to be tested.
It would be readily apparent to one of ordinary skill in the art that such an assay as described above would be useful in identifying tissue samples which contain farnesyl-protein transferase and quantitating the enzyme. Thus, potent inhibitor compounds of the instant invention may be used in an active site titration assay to determine the quantity of enzyme in the sample. A series of samples composed of aliquots of a tissue extract containing an unknown amount of farnesyl-protein transferase, an excess amount of a known substrate of FPTase (for example a tetrapeptide having a cysteine at the amine terminus) and farnesyl pyrophosphate are incubated for an appropriate period of time in the presence of varying concentrations of a compound of the instant invention. The concentration of a sufficiently potent inhibitor (i.e., one that has a Ki substantially smaller than the concentration of enzyme in the assay vessel) required to inhibit the enzymatic activity of the sample by 50% is approximately equal to half of the concentration of the enzyme in that particular sample.